AN ACTIVITY GEL ASSAY DETECTS A SINGLE, CATALYTICALLY ACTIVE HISTONE ACETYLTRANSFERASE SUBUNIT IN TETRAHYMENA MACRONUCLEI
AN ACTIVITY GEL ASSAY DETECTS A SINGLE, CATALYTICALLY ACTIVE HISTONE ACETYLTRANSFERASE SUBUNIT IN TETRAHYMENA MACRONUCLEI
复制标题
DOI：
10.1073/pnas.92.14.6364
复制
发表时间：
1995-07-03
影响因子：
11.1
通讯作者：
ALLIS, CD
中科院分区：
文献类型：
--
作者：
BROWNELL, JE;ALLIS, CD
纤毛原生动物嗜热四膜虫的大核具有与转录相关的组蛋白乙酰化密切相关的组蛋白乙酰转移酶活性。关于四膜虫中这种活性的多肽组成或来自任何细胞来源的任何可比活性，尚无明确的信息。乙酰转移酶活性凝胶测定法确定了该酶在四膜虫中的催化活性亚基。这种活性凝胶试验检测粗核提取物中单个55 kDa （p55）的多肽，以及柱纯化的部分，将[H-3]乙酰辅酶a中的[H-3]乙酸纳入核心组蛋白底物，直接聚合成SDS聚丙烯酰胺凝胶。通过所有色谱步骤，包括反相高效液相色谱法，p55精确地与乙酰转移酶活性结合。凝胶过滤层析表明该活性的分子质量为220 KDa，表明天然酶可能由四个相同的55 KDa亚基组成。此外，p55与二核小体和更大的多核小体密切相关，因此可以定义为a型组蛋白乙酰转移酶的组成部分。染色质相关。
Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation. Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source. An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena. This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates [H-3]acetate from [H-3] acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels. p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC. Gel-filtration chromatography of this activity indicates a molecular mass of 220 KDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa. Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A-i.e., chromatin associated.